Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Abstract

Alzheimer disease and several other neurodegenerative disorders are characterized by the accumulation of intraneuronal fibrils comprised of the protein Tau. Tau is normally a soluble protein that stabilizes microtubules, with splice isoforms that contain either three (3-R) or four (4-R) microtubule binding repeats. The formation of Tau fibrils is thought to result in neuronal damage, and inhibitors of Tau fibrillization may hold promise as therapeutic agents. The process of Tau fibrillization can be replicated in vitro, and a number of small molecules have been identified that inhibit Tau fibril formation. However, little is known about how these molecules affect Tau fibrillization. Here, we examined the mechanism by which the previously described aminothieno pyridazine (ATPZ) series of compounds inhibit Tau fibrillization. Active ATPZs were found to promote the oxidation of the two cysteine residues within 4-R Tau by a redox cycling mechanism, resulting in the formation of a disulfide-containing compact monomer that was refractory to fibrillization. Moreover, the ATPZs facilitated intermolecular disulfide formation between 3-R Tau monomers, leading to dimers that were capable of fibrillization. The ATPZs also caused cysteine oxidation in molecules unrelated to Tau. Interestingly, methylene blue, an inhibitor of Tau fibrillization under evaluation in Alzheimer disease clinical trials, caused a similar oxidation of cysteines in Tau and other molecules. These findings reveal that the ATPZs and methylene blue act by a mechanism that may affect their viability as potential therapeutic agents.

Active ATPZs cause the formation of a compact 4-R Tau monomer. 4-R Tau K18PL protein was incubated for 1 h at 37 °C in the absence (K18PL) or presence of the active ATPZ, CNDR-51348 (K18PL:51348). The Tau protein was also either treated with an inactive ATPZ, CNDR-51449 (K18PL:51449) or was pre-reduced (K18PL Red) or pre-oxidized (K18PL Ox). The samples were subsequently analyzed by SEC.

ATPZs promote intramolecular cysteine disulfide formation in 4-R Tau.A, K18PL was incubated for 1 h at 37 °C in the absence (−) or presence of CNDR-51348 (51348) or CNDR-51449 (51449) or was pre-reduced (R) or pre-oxidized (O). The samples were then analyzed for IAA-OG reactivity. Both the IAA-OG fluorescence and corresponding Coomassie blue staining are shown for each IAA-OG-treated sample after SDS-PAGE. B, K18PL, K18, or full-length 4-R Tau (T40) were incubated for 1 h at 37 °C in the absence (−) or presence of CNDR-51348 (51348) or CNDR-51449 (51449) or were pre-reduced (R) or pre-oxidized (O) followed by native gel electrophoresis. C, K18PL in which both cysteines were mutated to alanine (2xCA) was incubated for 1 h at 37 °C in the absence (2xCA) or presence of CNDR-51348 (2xCA:51348) followed by SEC analysis.

ATPZ-treated 3-R Tau undergoes intermolecular disulfide formation.A, the 3-R Tau K19 protein was incubated for 1 h at 37 °C in the absence (K19) or presence of CNDR-51348 (K19:51348) or CNDR-51449 (K19:51449) or was pre-reduced (K19 red) or pre-oxidized (K19 ox) before SEC analysis. B, K19 or full-length 3-R Tau (T39) were incubated for 1 h in the absence (−) or presence of CNDR-51348 (51348) or CNDR-51449 (51449) or were pre-reduced (R) or pre-oxidized (O) followed by native gel electrophoretic analysis. The bands corresponding to monomer (M) and dimer (D) are indicated.

4-R Tau proteins harboring single cysteine mutations form intermolecular disulfide bonds after ATPZ treatment.A, K18PL Tau protein containing the C291A mutation was incubated for 1 h at 37 °C in the absence (C291A) or presence of CNDR-51348 (C291A:51348) or CNDR-51449 (C291A:51449) or was pre-reduced (Reduced) or pre-oxidized (Oxidized) before SEC analysis. B, K18PL Tau protein containing the C322A mutation was incubated for 1 h at 37 °C in the absence (C322A) or presence of CNDR-51348 (C322A:51348) or CNDR-51449 (C322A:51449) or was pre-reduced (Reduced) or pre-oxidized (Oxidized) before SEC analysis.

ATPZs facilitate disulfide formation in thiol-containing molecules other than Tau.A, a 10-mer peptide containing two cysteine residues was incubated for 0.5 h at 37 °C in the presence or absence of CNDR-51348 or CNDR-51449, and the mixtures were subsequently analyzed by reversed-phase HPLC-MS. In addition, a pre-oxidized sample of the 10-mer was subjected to HPLC-MS analysis. Peaks with molecular masses corresponding to a peptide with oxidized cysteines and reduced cysteines eluted at 1.15 and 1.25 min, respectively. B, DTT was incubated in the absence (DTT) or presence of CNDR-51348 (DTT:51348) or was pre-oxidized (DTTox) before HPLC-MS analysis. The peaks eluting at 2.91 and 4.44–4.48 min have molecular masses corresponding to the reduced and oxidized forms of DTT, respectively, whose structures are depicted.

ATPZs facilitate molecular oxygen-mediated cysteine oxidation.A, HPLC-MS analysis of CNDR-51348 that was incubated for 1 h at 37 °C in the absence (51348) or presence of K18PL (51348:K18PL). The molecular mass of the compound was unchanged after oxidation of Tau. The two major m/z values (320 and 322) correspond to CNDR-51348 containing the naturally occurring 35Cl and 37Cl isotopes. B, CNDR-51348 or vehicle (water) were incubated for 1 h in the absence or presence of K18PL followed by the determination of peroxide concentrations in the incubation mixtures. C, K18PL was left untreated (−) or was pre-reduced (R). In addition, K18PL was treated with CNDR-51348 or with 20 μm (PLo) or 1 mm (PHi) hydrogen peroxide for 1 h at 37 °C in the presence or absence of catalase. The samples were subsequently analyzed by native gel electrophoresis or underwent reaction with IAA-OG followed by SDS-PAGE to evaluate the extent of cysteine oxidation. Both the IAA-OG fluorescence and corresponding Coomassie blue staining are shown for each IAA-OG-treated sample. D, fibrillization reactions were conducted with K18PL in the presence of CNDR-51348 (51348) or the presence of both CNDR-51348 and catalase (51348 + cat). E, shown is the proposed reaction scheme of ATPZ-mediated oxidation of Tau.

MB also facilitates formation of disulfide-stabilized 4-R Tau compact monomer as well as intermolecular disulfides in 3-R Tau and 4-R Tau with a mutated cysteine.A, K18PL was incubated for 1 h at 37 °C in the absence (K18PL) or presence of CNDR-51348 (K18PL:51348) or MB (K18PL:MB). The samples were subsequently analyzed by SEC. B, 3-R Tau K19 was incubated for 1 h in the absence (K19) or presence of CNDR-51348 (K19:51348) or MB (K19:MB) followed by SEC analyses. C, K18PL containing the C291A mutation was incubated for 1 h at 37 °C in the absence (C291A) or presence of CNDR-51348 (C291A:51348) or MB (C291A:MB).

MB induces disulfide formation in molecules other than Tau and does not inhibit 3-R Tau fibrillization.A, K18PL or K19 were incubated for 1 h at 37 °C in the absence (−) of presence of MB (MB) or were pre-reduced (Red) or pre-oxidized (Ox). The samples were then analyzed by native gel electrophoresis. In addition, T40 was incubated in the absence (−) or presence of MB (+MB) for 15 and 30 min followed by analysis by native gel electrophoresis. B, a 10-mer peptide containing two cysteine residues was incubated for 1 h at 37 °C in the absence (10-mer) or presence of MB (10-mer:MB) or was pre-oxidized (10-mer:Ox), and the mixtures were subsequently analyzed by reversed-phase HPLC-MS. Peaks with masses corresponding to a peptide with oxidized cysteines (m/z = 1152) and reduced cysteines (m/z = 1154) eluted at 1.08–1.09 and 1.20–1.21 min, respectively. The peak eluting at 1.30 min in the MB-treated sample is reduced methylene blue, and the shoulder on the peak eluting at 1.09 min is azure B, which is a demethylated form of MB that is found as a minor contaminant in MB preparations. C, K19 and K18 fibrillization reactions were incubated with increasing concentrations (in μm) of MB or oleocanthal (Oleo). After completion of the reactions, the samples were subjected to centrifugation to separate fibrillar Tau (pellet fraction (P)) from non-fibrillar Tau (supernatant fraction (S)). Both the pellet and supernatant fractions were analyzed by SDS-PAGE and Coomassie Blue staining. D, K18PL was left untreated (−) or was pre-reduced (R) or pre-oxidized (O). In addition, K18PL was treated with MB or with 20 μm (PLo) or 1 mm (PHi) hydrogen peroxide both in the presence and absence of catalase. The samples subsequently underwent reaction with IAA-OG followed by SDS-PAGE to evaluate the extent of cysteine oxidation. Both the IAA-OG fluorescence and corresponding Coomassie blue staining are shown for each IAA-OG-treated sample. In A and D, the vertical lines designate sites where non-pertinent gel lanes were removed and figures were subsequently spliced.

Tau cysteine oxidation by ATPZ or MB is inhibited by cellular concentrations of GSH. K18 (A) or K19 (B) were incubated for 1 h at 37 °C in the absence (−) or presence of CNDR-51348 (51348), CNDR-51449 (51449) or MB or was pre-reduced (R) or pre-oxidized (O). These reactions were conducted either in the absence (−GSH) or presence (+GSH) of 5 mm GSH. The samples were then analyzed by native gel electrophoresis.